CRISPR guides induce gene silencing in plants in the absence of Cas

Background RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. Results Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. Conclusions Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.

Cardiac-targeted PIASy Gene Silencing Mediates deSUMOylation of Caveolin-3 and Prevents Ischemia/reperfusion-induced Nav1.5 Down-regulation and Ventricular Arrhythmias

Background: Abnormal myocardial expression and function of Nav1.5 causes lethal ventricular arrhythmias during myocardial ischemia-reperfusion (I/R). PIASy mediated Caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to ligand Nav1.5. PIASy activity is increased after myocardial I/R, whether or not this may be attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias remains unclear. Methods: Using recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced by intraventricular injection of PIASy shRNA. Two weeks later, the hearts were subjected to I/R, and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurement. Results: We found that PIASy was upregulated by I/R, with increased SUMO2/3 modification of Cav-3, reduced membrane Nav1.5 density, and increased ventricular arrhythmia frequency. These effects were significantly reversed by PIASy silencing. In addition, PIASy silencing enhanced Cav-3 binding to Nav1.5 and prevented I/R-induced Nav1.5 re-localization. Using in vitro models of HEK293T cells and isolated adult rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R), this reserch further confirmed that PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of the SUMO Consensus Sites Lysine in Cav-3 (K38R or K144R) alters the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells. Conclusions: I/R-induced cardiac PIASy activation contributes to Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5- related ventricular arrhythmias. Cardiac-targeted PIASy gene silencing mediates deSUMOylation of Cav-3 and prevents I/R-induced Nav1.5 down-regulation and ventricular arrhythmias in rats, identifying PIASy as a potential therapeutic target for relevant life-threatening arrhythmias in patients with ischemic heart diseases.